Studies on the Myrosinase in Mustard Seed

In order to clarify whether the myrosinase containes only thioglucosidase, or thioglucosidase and sulfatase, chromatographic behaviors of the myrosinase on cellulose ion exchanger were studied. Myrosinase showed two peaks by the chromatography on TEAE-cellulose at pH 8.5, but the fractionation of the two enzymes was not accomplished. It was concluded that the two peaks was not ascribable to an artifact but the two closely resembled species of myrosinase existing in the sample solution. Chromatographic separation of the two enzymes on DEAE-cellulose resulted in failure. These results confirmed that the myrosinase was a single β-thioglucosidase originally.     

Studies on the Myrosinase in Mustard Seed

A characteristic effect of inorganic neutral salts on myrosinase was discovered. The salts containing monovalent anions had a remarkable inhibitory effect on the ascorbate-activated enzyme, but little on the non-activated enzyme. Such an effect was elucidated to be due to the anion. For the ascorbate-activated enzyme, a linear relation was obtained by plotting the logarithms of the enzymatic activity against the square roots of the ionic strength of the salts. Therefore, the effect of monovalent anions is ascribable to the ionic strength of the solvent. The Km values of the activated and non-activated enzyme were increased by the presence of the monovalent anion.     

Studies on Fungous Myrosinase

In order to obtain fungous myrosinase, Aspergillus sydowi IFO 4284 was cultured on a medium containing mustard seed extract for 2 weeks. Myrosinase in the broth was purified about 150 fold by precipitation with ammonium sulfate and chromatography on DEAE-cellulose and DEAE-Sephadex. Comparison of thioglucosidase and sulfatase activities of the myrosinase preparation using pH-activity, pH-stability and temperature-stability curves revealed no differences from each other. The chromatograms of the two activities on DEAE-Sephadex showed good agreement. Consequently, the myrosinase produced by Aspergillus sydowi was concluded to be a single β-thioglucosidase, not a mixture of thioglucosidase and sulfatase.

The effects of various reagents on Aspergillus sydowi myrosinase were studied.

The enzymatic activity was stimulated by cobalt (II), zinc (II) and magnesium ions and inhibited by mercury (II), iron (II) and copper (II) ions. However, metal-complexing agents, SH reagents and diisopropylfluorophosphate showed no effects on enzymatic activity. In contrast to plant myrosinase, this enzyme was neither activated nor inhibited by any concentrations of l-ascorbate. Glucose and salicin were competitive inhibitors for the enzyme. High concentrations of sodium chloride inhibited the enzyme.

From the inhibition modes of sugars and β-glucosides and from that of sodium chloride against the enzyme, a similarity of the enzyme to β-glucosidases was shown.

β-Glucosidase activity of fungous myrosinase was confirmed using p-nitrophenyl β-glucoside as a substrate. This activity was revealed to be due to the myrosinase itself, Experimental results indicated a resemblance of fungous myrosinase to β-glucosidases similar to plant myrosinase. The relationship between fungous and plant myrosinases to the β-glucosidases are discussed from the view of the substrate specificity of these enzymes. The conclusions are that distinction between plant and fungous myrosinases and the β-glucosidases are not as strict as previously thought, and the myrosinases should be considered β-glucosidases highly specialized for the hydrolysis of mustard oil glucoside.     

Possibility that Myrosinase is a Single Enzyme and Mechanism of Decomposition of Mustard Oil Glucoside by Myrosinase

Crystalline yeast phosphoglyceric acid mutase is composed of several components. Though they have the same crystalline forms, the differences are found among their specific activities. Hereupon, it is necessary to elucidate the enzymatic properties of these components. As the results, the basic properties of each crystalline component have been found to be the same.     

黑芥子酶研究进展

综述了黑芥子酶的研究进展,包括黑芥子酶催化硫代葡萄糖苷的水解产物对动植物和人体的作用;黑芥子酶的性质、提纯技术、活性测定、同工酶的基因家族;产黑芥子酶的植物、动物和微生物等。部分硫代葡萄糖苷可经黑芥子酶水解成有抗癌作用的异硫代氰酸盐。可通过测定葡萄糖的生成量或底物硫代葡萄糖苷浓度的变化来确定黑芥子酶的活性。十字花科等植物,以十字花科植物为食的拟步行虫、黄条跳甲、甘蓝蚜虫、小菜蛾、粉纹夜蛾等昆虫,啤酒酵母、交链孢霉属、茎点霉等真菌,以及多型拟杆菌等肠道细菌都有黑芥子酶。     

Molecular Markers for Seed Oil Content in Indian Mustard

With the objective of identifying molecular markers for the seed oil content in mustard (Brassica juncea), which is known to be quantitatively inherited, 94 F₆ generation recombinant inbred lines (RILs) developed from an intervarietal cross (Varuna × BEC144), were characterised. Oil content, determined by pulsed nuclear magnetic resonance technique, varied from 25.7% to 52.3% among the RILs. The frequency distribution of the trait was normal with mean 39.24 and standard deviation 4.52. Bulked segregant analysis of the RILs enabled fast and efficient screening of 219 random decamer primers for identification of informative primers. Single factor analysis of variance revealed significant association of oil content with three RAPD markers, viz., OPH11₁₃₅₀, OPJ06₁₂₀₀ and OPL05₆₀₀ which individually explained 6% of the trait variance. Molecular data indicated dispersion of desirable quantitative trait loci in the two parent varieties.     

Studies on the Activation Mechanism of the Myrosinase by L-Ascorbic Acid

The desmutagenic activity of various food components on C-nitro mutagens formed by the nitrite/sorbic acid reaction was assayed and several vegetable juices were found to be effective for eliminating the mutagenicity of the nitrite/sorbic acid system. Especially, the desmutagenic activity of pumpkin juice was investigated, and ascorbic acid, cysteine and other reducing compounds were found to be responsible for desmutagenic actions on 1,4-dinitro-2-methyl pyrrole, the main mutagen formed by the reaction of sorbic acid with sodium nitrite, by reduction of the conjugated C-nitro group to a C-amino group.     

植物芥子酶研究进展

芥子酶防御系统为白花菜目植物特有．由芥子酶及其底物硫代葡萄糖苷组成．芥子酶和硫代葡糖苷分别储藏在不同的细胞中,在受到病虫侵袭时,底物和酶相遇,硫代葡糖苷被降为有毒化合物,起防御作用．对植物芥子酶防御系统研究进展进行了综述,包括基因家族的结构、基因的表达调控、芥子酶的细胞定位、植物以外其它生物的芥子酶、硫代葡糖苷／芥子酶系统起源进化以及其可能功能等．     

麻疯树种子的研究进展

麻疯树（Jatropha curcas L．）为大戟科（Euphorbiaceae）麻疯树属半肉质小乔木或大灌木,具有很强的抗旱、耐贫瘠的特性。麻疯树的根、树皮、叶和种子均可人药。种子中主要含有脂肪类物质、蛋白质和萜类物质,其毒素为麻疯树毒蛋白和种子油。种仁中的含油量约为50％,可作为理想的生物柴油;毒蛋白、种子油及其他种子提取物可作为生物农药。关于麻疯树种子的发育、脱水行为及其调控研究较少。麻疯树是二种具有重要经济价值的战略资源。     

超干贮藏对芥兰种子生活力和活力的影响

1年的监测结果显示：芥兰种子开放贮藏时的生活力和活力下降最快,超干种子(含水量为4．91％、3．25％和2．84％)具有良好的耐藏性,其种子发芽率和简化活力指数增大,抗老化能力、超氧物歧化酶和过氧化氢酶的活性增强,膜进性降低,以2．84％含水量的芥兰种子贮藏效果为最好。     

Studies on Lotus Seed Protease

A protease from the lotus seed (Nelumbo nucifera Gaertn) was purified by acid-treatment, ammonium sulfate-fractionation, ethylalcohol-fractionation, TEAE-cellulose-treatment and Sephadex G-100 gel-filtration.

The enzyme was purified about 870-fold and was homogeneous in electrophoretic and ultracentrifugal analyses.

Purified lotus seed protease is an acid protease with a pH optimum at 3.8 toward urea-denatured casein. It is active for casein and hemoglobin. But other proteins such as edestin, zein, lotus seed globulin and soybean casein are slightly hydrolyzed and egg albumin is hardly hydrolyzed. This enzyme is most stable at pH 4.0 below 40°C. The enzyme is not a thiol protease, and its activity was completely inhibited by potassium permanganate, remarkably inhibited by sodium dodecylsulfate and accelerated by hydrogen peroxide.     

Studies on Lotus Seed Protease

Some physicochemical and enzymic properties of the purified lotus seed protease were investigated. The molecular weight determined from sedimentation-diffusion studies and by Sephadex G-100 gel-filtration was 36,800 and 35,500, respectively. The enzyme gave a typical ultraviolet spectrum of protein and its isoelectric point was found to be in a range of pH 3~4. The enzyme was stabilized at pH 6 in Tris-hydrochloric acid buffer by the addition of cupric ion. The inhibitory function of some reagents, especially that of potassium permanganate, and acetylation of the enzyme revealed that the tyosine residue in this enzyme protein might play some important role in the enzyme activity. Lotus seed protease was found to be fairly similar to pepsin in some properties.     

Myrosinase in Sinapis alba L.

Extracts from developing seeds, seedings, and different organsof mature plants of Sinapis alba L. were tested for the presenceof myrosinase. Three different patterns of isoenzymes were distinguishedby isoelectric focusing. One was found in developing seeds andseedlings, a second in mature leaves, stems, flowers, and podsand a third in roots. Sections of all the parts tested were examined for the presenceof myrosin cells by light microscopy. Developing seeds and seedlingsshowed a good correlation between the appearance of myrosinaseand the presence of myrosin cells. No myrosin cells were foundin root or flower parts although they showed myrosinase activity.     

Myrosinase in Raphanus sativus L.

A previous paper reported an investigation of myrosinase inthe seeds, seedlings and the different organs of mature plantsof Sinapis alba. The enzyme distribution was related to thehistological localization of myrosin cells. The present paperreports the situation for Raphanus sativus. Differences in myrosinasepatterns have been found but the two taxa are similar in thatthere is good evidence for the presence of the enzyme in myrosincells and in other non-specialized cells. Key words: Raphanus, Myrosinase, Isoelectric focusing, Myrosin cells     

Carbonic Anhydrase,Photosynthesis, and Seed Yield in Mustard Plants Treated with Phytohormones

The leaves of 30-d-old plants of Brassica juncea Czern & Coss cv. Varuna were sprayed with 10^–6 M aqueous solutions of indole-3-yl-acetic acid (IAA), gibberellic acid (GA₃), kinetin (KIN), and abscisic acid (ABA) or 10^–8 M of 28-homobrassinolide (HBR). All the phytohormones, except ABA, improved the vegetative growth and seed yield at harvest, compared with those sprayed with deionised water (control). HBR was most prominent in its effect, generating 32, 30, 36, 70, 25, and 29 % higher values for dry mass, chlorophyll content, carbonic anhydrase (E.C. 4.2.1.1) activity, and net photosynthetic rate in 60-d-old plants, pods per plant, and seed yield at harvest, over the control, respectively. The order of response to various hormones was HBR > GA₃ > IAA > KIN > control > ABA.     

Studies on the Alkylation of Choline Acetyltransferase by Choline Mustard Aziridinium Ion

Although a potent irreversible inhibitor of high-affinity choline transport in rat brain synaptosomes, choline mustard aziridinium ion (ChM Az) appeared to be a relatively weak inhibitor of choline acetyltransferase (ChAT) in rat brain homogenates, and evidence for irreversible binding of this compound to the enzyme had not been established. Accordingly, the irreversible inactivation of partially purified rat brain ChAT by ChM Az was studied. This compound is a rather weak inhibitor of the enzyme, with 50% inhibition of ChAT activity achieved following 30 min incubation at 37 degrees C with 0.6 mM ChM Az. This result indicates that although ChM Az has affinity for many nucleophiles there was little diluting effect of the inhibitor in the crude brain homogenate which could be attributed to such reactions (50% inhibition caused by 1.8 mM ChM Az following 10 min incubation). Although the initial binding of ChM Az to ChAT may be of a competitive nature, irreversible bond formation resulted. The time-dependent alkylation reaction conformed to pseudo-first-order kinetics with an observed forward rate constant (kobs) of 0.173 min-1; the half-time (t 1/2) for irreversible binding was about 4 min. The irreversible inactivation of ChAT by ChM Az would appear to be slower than the alkylation of high-affinity choline carriers in synaptosomes by this compound, and the relatively weak inhibitory action of ChM Az against either partially purified ChAT or ChAT activity in crude rat brain homogenates is in striking contrast to previous evidence that ChAT in intact synaptosomes was inhibited irreversibly by lower concentrations of the inhibitor.     

种子休眠的研究进展

种子休眠是植物在长期系统发育进程中获得的一种适应环境变化的特性。这种特性能够确保物种在恶劣的环境中存活, 减少同一物种中个体之间的竞争, 以及防止种子在不适宜的季节萌发。该文综述了种子休眠的类型、种子休眠的发育与连续群、种子休眠的调节、与休眠诱导、维持和释放有关的蛋白以及种子休眠的进化, 并提出了今后种子休眠研究的主要问题。     

种子休眠的研究进展

种子休眠是植物在长期系统发育进程中获得的一种适应环境变化的特性。这种特性能够确保物种在恶劣的环境中存活,减少同一物种中个体之间的竞争,以及防止种子在不适宜的季节萌发。该文综述了种子休眠的类型、种子休眠的发育与连续群、种子休眠的调节、与休眠诱导、维持和释放有关的蛋白以及种子休眠的进化,并提出了今后种子休眠研究的主要问题。     

Isolation and Characterization of Mustard (Sinapis alba L.) Seed Storage Proteins

A total storage protein fraction was prepared from mustard (Sinapis alba L.) seeds via isolated protein bodies and characterized by sedimentation, immunological, and electrophoretic techniques. Mustard seed storage protein consists of three fractions (1) a “legumin-like” 13-S complex composed of two pairs of disulfide-linked polypeptides (16.5 + 28.5 kDa and 19.5 + 34 kDa, respectively) and two single polypeptides (18 kDa and 26 kDa), (2) a “vicilin-like” 9-S complex composed of two glycoproteins (64 kDa and 77 kDa), and (3) two small polypeptides (10 kDa and 11 kDa) which probably represent the 1.7-S complex found in other Cruciferae. In contrast to related species, no glycosylated polypeptide was found in the 13-S complex. Immunological relationships were found between the paired polypeptides of the 13-S complex but not between polypeptides of the 13-S complex and polypeptides of the 9-S complex. Pulse-chase labeling and in vitro translation of polysomal RNA from young embryos demonstrated that the polypeptides of the 13-S complex originate from high molecular mass precursors, except for the 18 kDa polypeptide which appears to be synthesized in its final size. The amino-acid composition of the major polypeptides of the mustard storage protein is given.